Unlimited proliferation, infiltration and metastasis constitute the characteristics of a malignant tumor, and also the major reasons leading to failed treatment and death. Therefore, effective control of proliferation, infiltration and metastasis is the essential measure to improve prognosis and survival rate of tumor-bearing patients. In 1971, Folkman firstly pointed out that the growth of tumor relies on angiogenesis, which is the morphological basis for tumor proliferation and metastasis in that it not only provides nutrition required for tumor growth, but also transfers numerous tumor cells to the host's other organs and consequently results in metastasis. Most of malignant solid tumors, such as ovarian cancer, liver cancer, cervical cancer and breast cancer, are angiogenesis-dependent. As the new blood vessels formed through angiogenesis can on the one hand provide nutrition and oxygen for the tumor, and on the other hand act as important channel for tumor metastasis, inhibiting the process of angiogenesis is one of the most important measures to fight against cancer.
Integrins are a type of receptors widely found on the cell surface. They can induce adhesion of vascular endothelial cells and tumor cells, and facilitate angiogenesis and tumor metastasis by means of mediating interaction between intracelluar cytoskeletal proteins and extracellular matrix molecules. Currently, at least 8 integrins (α1β1, α2β1, α3β1, α6β1, α6β4, α5β1, αvβ3, αvβ5) have been found closely related to tumor angiogenesis, among them αvβ3 being the most important. Integrin αvβ3 is also called VN receptor. It is a transmembrane heterodimer glycoprotein consisting of an αv subunit (CD51, 150 kD) and a β3 subunit (CD61, 105 kD). Integrin αvβ3 is expressed in many cell types and can combine with a variety of ligands during multicellular activities; therefore, it is extensively involved in tumor angiogenesis, infiltration and metastasis. It has been found that integrin αvβ3 can recognize the sequence Arg-Gly-Asp (RGD) in its ligands, which means that an RGD-containing polypeptide can function as an integrin antagonist and inhibit angiogenesis by means of reducing the expression of adhered molecules on the cell surface and mediating intracellular signal transduction. This eventually slows down tumor growth and metastasis. In other words, integrin-targeting polypeptides can block intracellular signaling pathways downstream of the integrin and effectively inhibit tumor growth and metastasis by means of slowing down angiogenesis. These features provide integrin-targeting polypeptides very promising prospects in tumor treatment.
Currently, some integrin blockers have been developed out on the international market and are undergoing the phase II clinical trial. However, no such products are seen on the Chinese market. It is of great necessity to develop this type of drugs with China's independent intellectual property. The Chinese patent “Angiogenesis-inhibiting Polypeptides and Preparation and Application Thereof′ (ZL 200610039298.2) disclosed a series of polypeptides obtained through restructuring and modifying the 6-49 amino acids on the integrin sequence. In contrast with endostatin, the restructured polypeptides present higher in vivo activity and tumor-targeting performance. In this cited patent, several integrin inhibitors were introduced, two of which were Arg-Gly-Asp-Phe-Gln-Pro-Val-Leu-HiArg-Gly-Asp-Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser -Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val -Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala-Phe (SEQ ID NO: 4) (EDSM-1 for short) and Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn -Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala -Val-Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala-Phe-Gly-Gly-Gly-Gly-Ala-Cys-Arg-Gly-Asp-Cys-Phe-Cys (SEQ ID NO: 5) (EDSM-2 for short). Both of the sequences contain an integrin ligand sequence, consisting of Arg-Gly-Asp and Gly-Gly-Gly-Gly-Ala-Cys-Arg -Gly-Asp-Cys-Phe-Cys (SEQ ID NO: 6), and an angiogenesis-inhibiting sequence, namely, Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly -Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala-Phe (SEQ ID NO: 7). The above mentioned patent only conducted preliminary researches on cloning of polypeptides EDSM-1 and EDSM-2, construction of prokaryotically expressed vectors, and the application of EDSM-1 in treatment of liver cancer and gastric cancer. In contrast, the present invention, on the basis of further studies on the endostatin sequence, found out that the 6-48 amino acids on the integrin sequence demonstrate even better angiogenesis-inhibiting function, and then tried to modify this angiogenesis-inhibiting sequence, namely, Phe-Gln-Pro-Val-Leu -His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys -Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala (SEQ ID NO: 1), by adding an integrin ligand sequence (Arg-Gly-Asp-Gly-Gly-Gly-Gly) (SEQ ID NO: 14) to its N-terminal and C-terminal ends respectively. The modification results in two new integrin blockers, namely, polypeptide II (Arg-Gly-Asp-Gly-Gly-Gly-Gly-Phe-Gln-Pro-Val-Leu -His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys -Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala-Gly-Thr-Phe-Arg-Ala) (SEQ ID NO: 2) and polypeptide III (Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser -Gly-Gly-Met-Arg-Gly-Ile-Arg-Gly-Ala-Asp-Phe-Gln-Cys-Phe-Gln-Gln-Ala-Arg-Ala-Val-Gly-Leu-Ala -Gly-Thr-Phe-Arg-Ala-Gly-Gly-Gly-Gly-Arg-Gly-Asp) (SEQ ID NO: 3). These newly designed polypeptides, can on the one hand effectively bind to the integrin subtype specifically expressed by tumors as the RGD (Arg-Gly-Asp) sequence contained in the integrin ligand sequence can targetedly recognize the integrin, and on the other hand successfully inhibit tumor growth and metastasis by means of inhibiting the process of angiogenesisp, a function realized by the angiogenesis-inhibiting sequence contained in them. Therefore, both polypeptide II and polypeptide III demonstrate strong tumor-targeting performance and high integrin affinity simultaneously. It has been found that they exhibit desirable therapeutic effect on many types of tumors, which means they have a wide range of indications and entail great social benefits and market potential.
Rheumatoid arthritis (RA) is one of the commonest autoimmune inflammatory arthropathies and major causes for disability. It is a chronic, symmetrical multi-synovial arthritis of unknown etiology. The incidence rate of RA is about 0.5%-1.0% throughout the world and about 0.4% in China. It can attack people at any age, but the risk goes higher with the increase of age. In addition, RA is closely related to gender, and the male to female incidence ratio is 1:3. The female at the age of 45-55 are at the highest risk. The initial symptoms of RA are progressive pain and swelling in hands and wrists, particularly the swelling at the back of wrists. Though such symptoms can be relieved with common symptomatic treatments, they tend to reappear repeatedly due to irregular or underdosed medication. With the development of the disease, progressive stiffness of joints may appear early in the morning and usually lasts for more than one hour, meanwhile, some joint dysfunctions may also appear.
As is mentioned above, the etiology and pathogenesis of RA remain unknown, and its basic pathological manifestations include vasculitis and synovitis. When RA attacks, a layer of pannus forms on the synovial membrane due to angiogenesis, which consequently results in thickening of synovial membrane, increase of exudate, release of various cytokines, cartilage destruction and bone erosion. It can also affect surrounding tissues, such as muscular compartments, ligaments, tendon sheaths and muscles, and finally affect the stability of joints and lead to joint deformation and disability. The RA vasculitis may attack other organs throughout the body and manifests itself as a systemic disease.
Currently, drugs for RA treatment can be categorized into two types: symptom-controlling drugs and disease-controlling drugs. The symptom-controlling drugs can be further divided into 4 groups: 1. NSAIDs, long regarded as first-line anti-RA agents; there are more than dozens of NSAIDs available on the Chinese market; 2. glucocorticoids, very good anti-inflammatory agents; but they cannot significantly improve the symptoms and will lead to many serious side effects if being used alone for a long time. They can be used, however, in the short term in moderate dose before the slow-onset agents take effect, and would be necessary to form combined medication with the second-line agents in pulse therapy of RA flare-ups, particularly for those patients with extra-articular manifestations; 3. slow-onset, anti-rheumatic drugs, usually regarded as second-line agents, including antimalarials, sodium aurothiomalate (gold), penicillamine and sulfasalazine; they take effect considerably slowly, but have positive functions in improving the overall condition of RA patients. They are also called disease-modifying antirheumatic drugs (DMARDs); 4. immunosuppressants, including methotrexate, cyclophosphamide, azathioprine, tripterygium and sinomenine, etc.
Angiogenesis is one of the main histological characteristics of rheumatoid arthritis. It causes hyperplasia of synovial membrane and infiltration of inflammatory cells—the basis for the formation of pannus and final destruction of joints. Due to angiogenesis, newly-formed blood vessels invade the joint cartilage, which under healthy conditions contains no blood vessels. The invasion of blood vessels leads to the erosion of cartilage, pain and eventually deformation of the whole joint. Also due to angiogenesis, the thickness of patients' synovial membrane increases. Normally, the inner layer of synovial membrane in a health people contains only 1-2 layers of cells; however, it would increase to 4-10 layers (sometimes 20 layers) of cells when RA attacks. These increased cells are not only in great quantity, but also extremely active. They can secrete a large quantity of cytokines, signaling molecules and proteases, all of which accelerate the process of joint destruction. In addition, there are a large quantity of inflammatory cells, such as T cells, B cells and monocytes infiltrating in the synovial membrane of RA patients.
Under normal physiological conditions, angiogenesis is strictly regulated and is a necessary process particularly important for reproduction, fetal development, tissue repair and wound healing. It also takes place under many pathological conditions, including growth and metastasis of tumors, inflammatory disorders such as RA, psoriasis, osteoarthritis, inflammatory bowel disease (IBD, including Crohn's disease and ulcerative colitis) and others.
Integrin αvβ3 can recognize the Arg-Gly-Asp (RGD) sequence in ligand molecules and bind with a variety of ligands during the multicellular activities. These features enable it to participate in such tumor processes as angiogenesis, infiltration and metastasis as well as other physiological and pathological processes such as inflammation, wound healing, blood coagulation, etc. Therefore, polypeptides bearing the RGD sequence can function as integrin antagonists, and the RGD sequence can be adopted as a vector, targetedly delivering therapeutic polypeptides to the endothelium of newly generated blood vessels so that those angiogenesis-related diseases can be effectively treated. The RGD-bearing, angiogenesis-inhibiting polypeptides can not only block the pathways of oxygen and nutrients to the synovial membrane by inhibiting angiogenesis, but also directly lead to degeneration of blood vessels therein. Therefore, they can inhibit hyperplasia of synovial membrane of RA patients. In short, inhibition of angiogenesis is an essential step for treatment of RA, while the proliferation and migration of endothelial cells are two crucial mechanisms for angiogenesis.
The RGD (Arg-Gly-Asp) sequence contained in polypeptide II and polypeptide III enables these polypeptides to realize effective combination with integrins. Such a combination on the one hand inhibits the interaction between intracellular cytoskeletal proteins and extracellular matrix molecules, and on the other hand inhibits the cell-cell adhesion and the adhesion between cells and extracellular matrix. The intercellular signaling as well as signal transduction between cells and extracellular matrix are consequently blocked, and angiogenesis is therefore inhibited. Meanwhile, there also exists an angiogenesis-inhibiting sequence in the above mentioned polypeptides. Researchers have found that this sequence presents high effect in inhibiting angiogenesis—a feature bearing great significance in treatment of RA and other similar diseases. In short, Integrin-inhibiting polypeptides demonstrate desirable performance in treating RA by targeting at RA's angiogenesis process. This provides a new orientation for developing new anti-RA drugs.
The newly designed polypeptides disclosed in the present invention, namely, polypeptide II and polypeptide III, present strong integrin-targeting performance and high integrin affinity. The preliminary research has indicated that polypeptide II and polypeptide III can inhibit the proliferation and migration of endothelial cells as well as capillary formation; with flow cytometry analysis, it detected out that the function target of polypeptide II and polypeptide III is integrin αvβ3. Later research also found out that polypeptide II and polypeptide III can inhibit the formation of capillary structures of rats' aortic rings, and with the cell adhesion assay, it further proved that the function target of the polypeptides is integrin αvβ3, and concluded that the polypeptides can be adopted in treatment of RA. These conclusions broaden the indication range of said polypeptides and highlight their social benefits and market potential.